Methods for purifying viruses

ABSTRACT

The invention provides methods for purifying a virus from impurities in an aqueous medium.

BACKGROUND OF THE INVENTION

[0001] The cultivation and purification of viruses has becomeincreasingly important for gene therapy and vaccine development. Huygheet al. (Human Gene Therapy 6: 1403-1416 (1995)) disclosed a comparisonof several methods for purification of recombinant adenoviruses,including anion-exchange chromatography, size exclusion chromatography,immobilized zinc affinity chromatography, ultracentrifugation,concluding that the preferred process for purification of a recombinantadenovirus is nuclease treatment of a cell lysate, followed byfiltration through membrane filters, followed by DEAE chromatography,followed by zinc affinity chromatography.

[0002] In view of the ever-increasing need for purified viruses, forexample for use as viral vectors for gene therapy, improved methods ofpurification would be highly desired.

SUMMARY OF THE INVENTION

[0003] One aspect of the invention is a method for purification of avirus preparation comprising:

[0004] a) subjecting the virus preparation to anion-exchangechromatography, wherein the virus is eluted from an anion-exchangechromatographic medium; and

[0005] b) subjecting the virus product of step a to size exclusionchromatography, wherein the virus is eluted from a size exclusionchromatographic medium. The virus preparation can be a cell lysate,which can be filtered before step A. The virus can be recombinantadenovirus, such as ACN53 (disclosed in WO 95/11984).

[0006] The anion exchange medium can comprise diethylaminoethyl groupson a cross-linked agarose, cellulose, polyacrylamide or polystyrenebackbone, such as FRACTOGEL™-DEAE. The size-exclusion medium cancomprise a cross-linked polysaccharide, and may be a composite ofcross-linked agarose and dextran. An exemplary size exclusion medium isSuperdex-200. The anion exchange chromatographic medium can beextensively washed before application of the virus preparation.

[0007] The size-exclusion medium can be provided in a column prepared asa salt gradient decreasing in ionic strength from the top of the columntowards the bottom, the top of the column having a buffer having anionic strength substantially identical to that of the product of step a.

[0008] A further aspect of the invention is a virus purified by themethod of claim 1.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0009] The present invention relates to the purification of a virus,which may for example have been produced by cultivation in a cellularhost and then liberated by lysis of the cells and separation fromcellular debris. The term “virus” includes wild type, mutant, andrecombinant viruses, especially adenoviral vectors for expression ofheterologous nucleic acid sequences.

[0010] The embodiments of the invention fall into the general strategyof adsorption chromatography of a virus preparation followed by sizeexclusion chromatography. Typically the anion exchange chromatography iscarried out on an anion-exchange resin consisting of basic groups inside chains attached to a macromolecular backbone. The basic groups arepreferably substituted amino groups, in particulardiloweralkylaminoalkyl groups where each lower alkyl group has 1 to 4,preferably 2, carbon atoms, and each alkyl group has 2 to 4, preferably2, carbon atoms. The backbone can be composed of silica or an organicmatrix, for example cross-linked agarose, cellulose, polyacrylamide orpolystyrene; it is particularly preferred to use an anion exchange resinconsisting of dimethylaminoethyl groups (DMAE groups) or especially ofdiethylaminoethyl groups (DEAE groups) on a cross-linked agarosebackbone; especially preferred resins of the DEAE type are those soldunder the trade name “DEAE-Fractogel”, e.g., “FRACTOGEL™ EMD DEAE-650M”,and “FRACTOGEL™ AE. In some embodiments of the invention, the “backbone”can be a solid support such as a bead.

[0011] The anion-exchange-resin is preferably washed extensively beforeloading the virus preparation to remove preservatives such as sodiumazide and ethanol, and other extraneous materials, by washing the columnwith about 5 to 10 column volumes of a basic solution such as 50 mMNaOH/1 M NaCl, followed by about 5 to 10 column volumes of aneutralizing solution such as 50 mM HCl/1 M NaCl, followed by about 5 to30 volumes of loading and/or elution buffers. Optionally, the column iswashed with a buffer of lower salt concentration than the loading and/orelution buffer before washing with loading and/or elution buffer.

[0012] Typically, a preparation of virus such as a cell lysate is loadedonto the chromatographic medium in a buffered solution of about pH7.0-8.5, with a salt concentration of about 100-360 mM. The salt istypically NaCl. In some embodiments other buffers such as phosphate orTris are used. Contaminants can be preferentially eluted by washing thecolumn with a buffer at a salt concentration of about 250-380 mM. Thevirus can then be eluted by a solution with a salt concentration ofabout 360-600 mM. The salt is typically NaCl. Typically, about 5 to 50,more preferably about 30 volumes of buffer are used to elute the virus.Fractions may be collected and assayed for the presence of virus bymeasuring the A₂₆₀ or A₂₈₀ and pooling peak fractions; alternatively,the eluant containing the A₂₆₀ or A₂₈₀ peak may be collected in a singlefraction. This single A₂₆₀ or A₂₈₀ fraction or pooled fractions in theeluant containing the virus are termed “anion-exchange pool” herein.

[0013] In the size-exclusion chromatography step, molecules areseparated according to size in a bed packed with an inert porous medium,especially an inert gel medium, which is preferably a composite ofcross-linked polysaccharides, e.g., cross-linked agarose and dextran inthe form of spherical beads. Molecules larger than the largest pores inthe swollen gel beads do not enter the gel beads and therefore movethrough the chromatographic bed fastest. Smaller molecules, which enterthe gel beads to varying extent depending on their size and shape, areretarded in their passage through the bed. Molecules are thus generallyeluted in the order of decreasing molecular size. Viruses, because oftheir large size, generally elute in the void volume. For example,adenoviruses have a diameter of approximately 80 nm. Media appropriatefor size-exclusion chromatography of adenoviruses include but are notlimited to such resins as G6000PWXL (TosoHaas); SB-806 (Alltech);Sephacryl S-400 HR, Sephacryl S-500 HR, Sephacryl S-1000 SF, SephadexG-200, Sepharose CL-2B; Superdex 200 prep grade, Superose 6 prep grade(Pharmacia); TSK 6000PWXL (Bodman), and Ultrahydrogel 2000 (Waters).

[0014] “Size-exclusion” chromatography as used herein is intended toinclude gel filtration chromatography. A particularly preferredsize-exclusion medium is that sold under the trade name “Superdex 200”;see the Pharmacia Catalog, 1996, pages 338-339, code no. 17-1043-01(bulk), or 17-1069-01 or 17-1071-01 (pre-packed columns). Since a groupseparation of virus from impurities of lower molecular weight isachieved, the loading volume of starting materials from theanion-exchange pool can be relatively large, e.g., up to 20%, morepreferably 15%, of the bed volume.

[0015] Exemplary materials for the practice of the anion-exchange andsize exclusion chromatographic steps of the invention are provided inTable I. Exemplary variables and controls are provided in Tables II andIII. TABLE I Exemplary Materials Used In Anion-Exchange and SizeExclusion Chromatography Purification Step Procedure Solution UsedDEAE-Fractogel Salt Adjustment 4 M NaCl Equilibration 265 mM NaCl, 2 mMMgCl₂, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer A) 50mM NaOH, 1M NaCl 100 mM HCl, 1M NaCl Wash 1 265 mM NaCl, 2 mM MgCl₂, 2%(w/v) sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer A) Wash 2 265 mMNaCl, 2 mM MgCl₂, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5(Buffer A) 600 mM NaCl, 2 mM MgCl₂, 2% (w/v) sucrose, 50 mM sodiumphosphate at pH 7.5 (Buffer B) Elution 265 mM NaCl, 2 mM MgCl₂, 2% (w/v)sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer A) 600 mM NaCl, 2 mMMgCl₂, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer B)Superdex 200 Equilibration 130 mM NaCl, 2 mM MgCl₂, 2% (w/v) sucrose, 50mM sodium phosphate at pH 7.5 (Buffer C) Elution 130 mM NaCl, 2 mMMgCl₂, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer C)

[0016] TABLE II In-process Control and Operating Variables for theAnion-Exchange Chromatography Purification Procedure VariableRecommended Value All procedures Temperature 4-12° C. Equilibration Flowrate <5 cm/min. 1) NaOH/NaCl Volume 5 column volumes pH of effluent 12.02) HCl/NaCl Volume 6 column volumes pH of effluent 8.0 3) Buffer AVolume 10 column volumes pH of effluent equivalent to Buffer A_0.2 pHLoad Flow rate <5 cm/min Conductivity of the feed 20-30 mS Wash 1:Buffer A Flow rate <5 cm/min Volume 4 column volumes Wash 2: Buffer A/Flow rate <5 cm/min Buffer B Volume 8 column volumes Elution: Buffer A/Flow rate <5 cm/min Buffer B Volume 30 column volumes Fraction SelectionA₂₈₀ >>background

[0017] TABLE III In-Process Control and Operating Variables for theSize-Exclusion Chromatography Purification Procedure VariableRecommended Value All procedures Temperature 4-12° C. Equilibration:Buffer C Flow rate <1 cm/min Volume 1 column volume pH of Effluentequivalent to Buffer C_0.2 pH Load Flow Rate <1 cm/min Volume 0.2 columnvolumes Concentration 30 A₂₈₀/mL Elution: Buffer C: Flow rate <1 cm/minVolume 1 column volume Fraction Selection A₂₈₀ >>background

[0018] In an embodiment of the invention, the virus is loaded from theanion-exchange pool onto a size-exclusion column. In some embodiments,the column is prepared with a salt gradient decreasing in ionic strengthfrom the top towards the bottom of the column. After loading, the virusmoves down through the salt gradient (since the virus is preferentiallynot adsorbed by the resin) and the gentle change in ionic strengthavoids damage to the virus. After overtaking the salt gradient, thevirus is eluted in a low-salt (e.g. 0-200 mM NaCl) buffer. Such low saltbuffers include, but are not limited to, formulations for long termstorage or administration to patients.

[0019] In some embodiments, glycerol is added to the chromatographicbuffers, such as elution buffer, or to the pooled fractions containingvirus. Typically the glycerol is present in a final concentration of5-20%, more typically 10%. Thus, in some embodiments, glycerol ispresent in all solutions throughout the process. In further embodiments,other excipients, such as about 2-16% sucrose, may be used in place ofthe glycerol.

[0020] In a preferred embodiment, the size exclusion chromatographycolumn is equilibrated with buffer at low salt concentration, e.g.,about 100 to 150 mM, especially about 130 mM NaCl. Just prior to loadingthe feed, a salt gradient is loaded, equivalent to a moderate fractionof the bed volume, e.g. 10 to 20%, preferably about 15%, from low saltconcentration (about 130 mM NaCl) to the higher salt concentration ofthe feed (e.g., 400 to 450 mM NaCl, especially about 420 nM NaCl).

[0021] A simple test is performed to determine the quality of theDEAE-Fractogel pool and consequently whether a salt gradient should beused. This test depends on the constancy of the A₃₂₀/A₂₆₀ ratio of theDEAE-Fractogel pool diluted with an appropriate pH 7.5 buffer over aperiod of a few minutes (e.g., 5 minutes). An appropriate bufferconsists of 50 mM sodium phosphate, pH 7.5, 2 mM MgCl₂, 2% sucrose, noNaCl. If the A₃₂₀/A₂₆₀ ratio remains substantially constant over a5-minute period (e.g., if it increases by no more than 0.04), then thatsample is suitable for either isocratic or salt-gradient size-exclusionchromatography. If the ratio of A₃₂₀/A₂₆₀ increases more than about 0.04during that period, then that DEAE-Fractogel pool is preferablyperformed on salt-gradient size-exclusion chromatography to improve theyield. Table IV provides exemplary materials and protocols for the useof salt gradient size exclusion chromatography. TABLE IV Exemplarymaterials and protocols for the use of salt gradient size exclusionchromatography Step Procedure Typical Range Preferred values Step (I)Equilibrate the 100 to 150 mM 130 mM NaCl column with the NaCl low saltbuffer Step (ii) Generate salt (100-150) to 130 to 450 mM gradient attop (400-500) NaCl; of column mM NaCl; 10 to 15% bed volume 20% bedvolume Step (iii) Load the DEAE- 10 to 20% bed 15% bed volume pool ontothe volume column. Step (iv) Elute the adenovirus 400 to 500 mM 450 mMNaCl with high-salt NaCl solution Step (v) Complete the 400 to 500 mM450 mM NaCl elution of the NaCl Virus.

[0022] The purification method of the present invention is suitable forscaling-up (or scaling down) and for large-scale containment. Suitableprocedures and guidelines well-known in the art can be used and followedto control the virus and prevent biohazardous situations: see, e.g.,“Biosafety in Microbiological and Biomedical Laboratories”, 3rd Edition,edited by Richman and McKinney, U.S. Department of Health and HumanServices, published by the Center for Disease Control and the NationalInstitute of Health, Washington, D.C., U.S. Government Printing Office,May 1993.

[0023] The methods of the instant invention are amenable to a wide rangeof viruses, including but not limited to adenoviruses, pox viruses,iridoviruses, herpes viruses, papovaviruses, paramyxoviruses,orthomyxoviruses, retroviruses, and rotaviruses. The viruses arepreferably recombinant viruses, but can include clinical isolates,attenuated vaccine strains, and so on.

[0024] Thus, for example, an exemplary recombinant adenovirus is thatcan be purified by the method of the invention is ACN53, which isdisclosed in PCT patent application no. WO 95/11984.

[0025] In the first step, the ACN53 adenoviral vector is purified byanion exchange chromatography on a DEAE-column. For this, the virus istypically propagated in 293 kidney cells, harvested, and subjected toconcentration and ultrafiltration. The concentrate is frozen and storedat about −20_C until use. Frozen concentrate is thawed, clarified byfiltration through a 0.45_m filter, the conductivity of the preparationadjusted to about 250-360 mM NaCl, and subjected to DEAE chromatography.Solutions used in the chromatography are listed in Table 1.

[0026] In the second step, the ACN53 adenoviral vector is purified bysize-exclusion chromatography on a Superdex-200 column. Selectedfractions containing virus are identified by A₂₆₀ or A₂₈₀ and pooled.The pooled fractions constitute the purified bulk ACN53 adenoviralvector which is then filtered sterile through a 0.2_m filter and storedat about − 20_C.

[0027] The virus in the DEAE-Fractogel pool may be unstable due to thepresence of a high concentration of salt (about 420 mM NaCl). It ispreferably processed immediately or stored at 4-12_C for not more thanabout 24 hours.

[0028] Fractions of the elution profile showing the ACN53 adenoviralvector peak as determined by A₂₆₀ or A₂₈₀ are pooled for furtherprocessing. The size-exclusion pool is filtered through a 0.2_m filter.This filtrate, the final purified bulk ACN53 adenoviral vector, is thentransferred into sterile plastic bottles (e.g. Teflon) and stored atabout −20° C. The in-process controls for this step are listed in TableIII.

[0029] The increase in purity of the ACN53 adenoviral vector at eachstep of the purification method can be followed by Resource Q HPLC (seeHuyghe et al., Human Gene Therapy, Vol. 6 (November 1995), pp. 1403-1416at p. 1405). The quality of the virus in the first and secondchromatographic pools is also monitored by spectroscopic methods. Thecharacteristic ratio of A₂₆₀/A₂₈₀ is 1.23-1.31:1 for final purifiedvirus. The light scattering which results from the high molecular weightof the virus is derived from the ratio of A₃₂₀/A₂₆₀ nm and is also usedto monitor the chromatographic pools. Purified, free virus particlesdisplay a light scattering ratio of about 0.22-0.30:1.

[0030] The following Examples serve to illustrate the present invention.The selected vectors and hosts and other materials, the concentration ofreagents, the temperatures, and the values of other variables are onlyto exemplify how the present invention may be carried out, and are notto be considered limitations thereof.

EXPERIMENTAL EXAMPLES

[0031] A. Small Scale Purification of Adenovirus

[0032] (1) Anion-Exchange Chromatography (DEAE-Fractogel)

[0033] A DEAE-EMD Fractogel 650M column (E. Merck), 5×18 cm., waspre-equilibrated with 5 bed volumes (B.V.) of 0.5 M NaOH/1 M NaClfollowed by 6 B.V. of 0.1 M HCl/1 M NaCl, and then by 20 B.V. of BufferA (265 mM NaCl, 2 mM MgCl₂, 2% (w/v) sucrose, 50 mM sodium phosphate atpH 7.5) at a linear flow rate of 2 cm/min. The feed for this column wasderived from 2 liters of frozen crude virus solution, which was thawed,microfiltered through a 0.45_membrane, and adjusted with a small volumeof 4 M NaCl to a conductivity equal to that of Buffer A. The feed wasloaded onto the column at a linear flow rate of 1 cm/min. The column waswashed with 4 B.V. of Buffer A. The column was then washed with 8 B.V.of 94% Buffer A/6% Buffer B (identical to Buffer A except that NaCl was600 mM). The column was eluted with 30 B.V. of a linear gradient from94% Buffer A/6% Buffer B to 100% Buffer B. Fractions containingsubstantial virus were pooled to form the feed (“DEAE pool”) for thefollowing column.

[0034] (2) Isocratic Size-Exclusion Chromatography (Superdex-200)

[0035] Size exclusion chromatography was performed on a Superdex-200column (Pharmacia), 5×73 cm, pre-equilibrated with 0.5 B.V. 0.5 M NaOH,1 B.V. of H₂O, and 2 B.V. of Buffer C (130 mM NaCl, 2 mM MgCl₂, 2% (w/v)sucrose, 50 mM sodium phosphate at pH 7.5) at a linear flow rate of 0.6cm/min. The feed consisting of 220 ml of DEAE pool was loaded onto thecolumn. ACN53 was eluted with Buffer C at a linear flow rate of 0.6cm/min. Fractions with substantial virus were pooled, passed through a0.2_microfilter and stored. The virus concentrate can be stored at lowtemperature, e.g., at 0-10_C, preferably at about 4_C, or if the volumeis small, e.g., less than about 50 ml, frozen at −80_C.

[0036] (3) Salt-Gradient Size-Exclusion Chromatography

[0037] (a). Salt Dilution Test

[0038] The DEAE-Fractogel pool (0.4 ml) was mixed with of a bufferconsisting of 50 mM sodium phosphate, pH 7.5, 2 mM MgCl₂, 2% sucrose, noNaCl (0.8 ml) and immediately placed in a quartz cuvette and measuredfor absorbance at 260 and 320 nm on a UV spectrometer equipped with aphotodiode array. Without removal of the sample from the cuvette, thereading was repeated at 1-2 minute intervals over a 5-minute period. Ifthe ratio of A₃₂₀/A₂₆₀ was substantially constant during that period,then that DEAE-Fractogel pool was suitable for either isocratic orsalt-gradient size-exclusion chromatography. If the ratio of A₃₂₀/A₂₆₀increased more than about 4% during that period, then thatDEAE-Fractogel pool required salt-gradient size-exclusion chromatographyto improve the yield.

[0039] (b) Salt-Gradient Size-Exclusion Chromatography

[0040] A salt-gradient chromatography was performed on a Superdex-200column, 2.6 cm×60 cm, pre-equilibrated with 0.5 B.V. 0.5 M NaOH, 1 B.V.of H₂O, and 2 B.V. of Buffer C (20 mM sodium phosphate, pH 8.0, 130 mMNaCl, 2mM MgCl₂, 2% sucrose). Immediately prior to loading the DEAEpool, a linear gradient from 100% Buffer C to 100% Buffer D (20 mMsodium phosphate, pH 8.0, 420 mM NaCl, 2 mM MgCl₂, 2% sucrose) of 0.15B.V. (48 ml) was applied to the Superdex-200 column. The feed consistingof 20 ml of a DEAE pool that had failed the above test was then loadedonto the column and eluted with Buffer D at a linear flow rate of 0.6cm/min. Fractions with substantial virus eluting in or near the voidvolume were pooled, passed through a sterilizing filter and stored at−80_C. The step yield was 60% and the A₃₂₀/A₂₆₀ ratio was 0.24:1.

[0041] B. Large-Scale Purification of Adenovirus

[0042] (1) Anion Exchange Chromatography

[0043] The frozen vial concentrate from the fermentation and recoverystep was thawed and filtered through a 0.45_m hydrophilic Duraporemembrane in a Millipore 10″ Opticap capsule. The filtrate was collectedin a closed tank. To minimize losses, the filter cartridge was washedwith about 1.5 L of buffer J-1 (50 mM sodium phosphate pH 7.5, 265 mMsodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose) supplementedwith 5.4% (w/w) of solution J-3 (4 M sodium chloride). The saltconcentration of the filtrate was adjusted by adding 5.4% (w/v) ofsolution J-3 (4 M sodium chloride). This feed solution was then appliedto a Fractogel EMD DEAE-650 M column (7 cm diameter, 14.8 cm bed height,570 ml bed volume) pre-equilibrated with buffer J-1 (50 mM sodiumphosphate pH 7.5, 265 mM sodium chloride, 2 mM magnesium chloride, 2%(w/v) sucrose). The Adenovirus binds to the anion exchange resin,whereas the majority of media and host cell impurities pass through thecolumn in the spent charge. The column was initially washed with 4 bedvolumes of buffer J-1 followed by a second isocratic wash of 8 bedvolumes of 94% buffer J-1 and 6% buffer J-2 (50 mM sodium phosphate pH7.5, 600 mM sodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose)to remove additional impurities. The virus was eluted from the columnwith a 30 bed volume linear gradient from 6% to 100% buffer J-2. Theadenovirus peak of the elution profile as determined by A₂₈₀ wascollected and pooled for further processing. The in-process controls andoperating parameters for the anion exchange chromatography step aresummarized in Table V. TABLE V In-Process Control and OperatingParameters for the Anion-Exchange Chromatography Column size: 7 cmdiameter, 14.8 cm bed height, 570 ml bed volume Purification ProcedureParameter Value All Procedures Temperature 3 to 9_C. Equilibration FlowRate 4 cm/min Volume 22 Column Volumes pH of Effluent Equal to BufferJ-1 (7.4) Load Flow Rate 1 cm/min Conductivity of the Feed 28.2 mS Wash1 Flow Rate 2 cm/min Volume 4 Column Volumes Wash 2 Flow Rate 2 cm/minVolume 8 Column Volumes Elution Flow Rate 2 cm/min Volume 30 ColumnVolumes Fraction Selection A₂₈₀ Peak area

[0044] (2) Size Exclusion Chromatography

[0045] The DEAE-Pool was applied immediately to a Superdex-200 sizeexclusion column (14 cm diameter, 77 cm bed height, 11.9 L bed volume)pre-equilibrated with buffer K-1 (20 mM sodium phosphate pH 8.0, 100 mMsodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose). The columnwas eluted with buffer K-1. The adenovirus peak of the elution profileas determined by A₂₈₀ was collected and pooled. This chromatography stepachieved a buffer exchange and separation of low molecular weightimpurities from the adenovirus product. The in-process controls andoperating parameters for the size exclusion chromatography step aresummarized in Table VI. TABLE VI In-Process Control and OperatingParameters for the Size Exclusion Chromatography Column size: 14 cmdiameter, 77 cm bed height, 11.9 L bed volume Purification ProcedureParameter Value All Procedures Temperature 3 to 9_C. Equilibration FlowRate 0.43 cm/min Volume 2.3 Column Volume Load Flow Rate 0.43 cm/minVolume _0.09 Column Volumes Concentration 2.7 A₂₈₀/mL Elution Flow Rate0.43 cm.min Volume 1.5 Column Volume Fraction Selection A₂₈₀ Peak area

[0046] (3) Final 0.2_m Filtration

[0047] The Superdex 200-pool was filtered through a 0.2_m hydrophilicDurapore membrane (Stericup, Millipore) at 2 to 15_C. This step wascarried out under sterile conditions in a biosafety cabinet. Becauseseveral filtration devices were used, the individual filtrates werepooled and then aliquoted into autoclaved containers. The containers ofbulk drug substance in solution were frozen in a dry ice/ethanol bathand stored at about −20 C.

[0048] All publications and patent applications cited herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

[0049] Modifications and variations of this invention will be apparentto those skilled in the art. The specific embodiments described hereinare offered by way of example only, and the invention is not to beconstrued as limited thereby.

What is claimed is:
 1. A method for purification of a virus preparationcomprising: a) subjecting the virus preparation to anion-exchangechromatography, wherein the virus is eluted from an anion-exchangechromatographic medium; and b) subjecting the virus product of step A tosize exclusion chromatography, wherein the virus is eluted from a sizeexclusion chromatographic medium.
 2. The method of claim 1 , wherein thevirus preparation is a cell lysate.
 3. The method of claim 2 , whereinthe cell lysate is filtered before step a.
 4. The method of claim 1 ,wherein the virus is a recombinant adenovirus.
 5. The method of claim 1, wherein the anion-exchange medium is FRACTOGEL™-DEAE.
 6. The method ofclaim 1 , wherein the size exclusion medium is Superdex-200.
 7. Themethod of claim 1 , wherein the size-exclusion medium is provided in acolumn prepared as a salt gradient decreasing in ionic strength from thetop of the column towards the bottom, the top of the column having abuffer having an ionic strength substantially identical to that of theproduct of step a.
 8. The method of claim 1 wherein the anion exchangemedium comprises diethylaminoethyl groups on a cross-linked agarose,cellulose, polyacrylamide or polystyrene backbone.
 9. The method ofclaim 1 , wherein the size-exclusion medium comprises a cross-linkedpolysaccharide.
 10. The method of claim 9 , wherein the cross-linkedpolysaccharide is a composite of cross-linked agarose and dextran. 17.The method of claim 1 wherein the virus is ACN53.
 18. The method ofclaim 1 , wherein the anion exchange chromatographic medium isextensively washed before application of the virus preparation.
 19. Avirus purified by the method of claim 1 .